Epidermolysis bullosa (EB) is a group of rare inherited skin blistering diseases that result in severe blistering and scaring. Some of the variants of EB sentence those afflicted to a life of severe pain and disability and even early death. Although the genetic defects that cause these devastating diseases have been known for almost 2 decades, current therapy for EB is limited to wound care. Since the epidermis is continuously renewed by stem cells in the proliferative basal layer, a permanent corrective therapy for EB must target the stem cell population. To date, no one has reported the successful use of homologous recombination technology to correct a defective gene in human keratinocyte stem cells isolated from an EB patient. The only successful clinical trial for an inherited skin blistering disease utilized a retroviral vector to restore expression of the missing protein, and that trial was placed on hold because of safety concerns with the use of retroviral vectors. For this reason, we believe that alternative strategies must be explored. Therefore, we are proposing to develop stem-cell based therapies for EB using autologous induced pluripotent stem cells (iPSC) derived from skin cells harvested from the same EB patient. The generation of patient-specific iPSC would not only potentially avoid the complication of immune rejection, but also provide a source of rejuvenated adult stem cells that are most likely exhausted as a result of unsuccessful attempts to repair blistered tissues. Prior to testing an iPSC-based stem cell therapy for EB in humans, it is desirable to utilize a pre-clinical animal model to determine the safety and efficacy of these approaches. We have previously generated an inducible mouse model that mimics the most severe form of epidermolysis bullosa simplex, Dowling-Meara (EBS-DM), at the genetic level. Using this model, we have obtained very compelling data documenting that EBS keratinocyte stem cells exhibit a growth disadvantage compared to wild-type keratinocyte stem cells. These results suggest that EBS is an example of an inherited skin disease where there would be a natural selection for genetically corrected iPSC-derived keratinocytes if they were seeded into areas prone to blistering. For this reason, and the fact that genetically corrected iPSC-derived keratinocytes would not be rejected by the patient's immune system, since EBS is a dominant form of EB, we believe that EBS is an ideal model for generating proof of concept data supporting the use of iPSC for the treatment of EB. Patients with EBS-DM develop lesions in the skin and the oral mucosal epithelia; therefore, both local and systemic therapy will be required to treat these patients. Dr. Jakub Tolar was the first to perform clinical trials using systemically delivered allogeneic bone marrow (BM)-derived cells in the treatment of RDEB patients, and for that reason, he was recruited as a PI on this multi-PI application. Although Dr. Tolar's initial clinical trials were very promising, there are safety concerns with allogeneic transplants, such as toxicity to chemotherapy required for conditioning before transplantation, and susceptibility to infections due to the necessity for immunosuppressive therapy to prevent rejection of allogeneic transplants. Unfortunately, 2 of the 7 RDEB children included in this initial trial died due to these complications. To avoid the complications of allogeneic transplants, we are proposing to generate iPSC from EBS-DM patients, and use zinc-finger nuclease (ZFN)-mediated genome editing to inactivate the mutant keratin 14 (KRT14) allele, which is defective in the majority of EBS-DM patients. We are proposing to use these corrected iPSC to generate keratinocytes to repair the skin by grafting, and mesenchymal cells to systemically repair lesions in the oral cavity. We have made substantial progress in overcoming some of the obstacles that will be required before iPSC can be used safely in the clinic, such as developing a non-viral method for reprogramming and efficient protocols for differentiating iPSC into keratinocytes and mesenchymal cells, but other safety issues remain. In this application, we are proposing to use novel models to address the remaining issues of histocompatibility, tumorigenicity, and genetic stability of iPSC-derived cells We have also designed a ZFN-mediated strategy to inactivate a 'hot spot mutation responsible for ~70% of the EBS-DM cases. If the studies outlined in this application are successful, these pre-clinical data will pave the way for approval of iPSC-based clinical trials for not only other forms of EB, but also other inherited skin disorders. PUBLIC HEALTH RELEVANCE: Epidermolysis bullosa (EB) is a group of rare inherited skin blistering diseases that result in severe blistering and scaring, and in some cases, early death. Current therapy for EB is limited to wound care. We are proposing to develop stem-cell based therapies for EB using autologous induced pluripotent stem cells (iPSC) derived from skin cells harvested from the same EB patient. If the studies outlined in this application are successful, these pre-clinical data will pave the way for approval of iPSC-based clinical trials for EB patients.